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Dove, Brian Kenneth. "Infectious bronchitis virus defective RNA studies." Thesis, University of Reading, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250714.
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Bhattacharjee, Partha Sarathi. "Enterotropism and persistence of avian infectious bronchitis virus in chickens." Thesis, University of Liverpool, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240459.
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Ambali, Abdul-Ganiyu. "Pathogenesis of an enterotropic variant of avian infectious bronchitis virus." Thesis, University of Liverpool, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.232935.
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Foltz, Jeffrey Andrew. "Characterization of infectious bronchitis virus isolates discovered during the 2004 avian influenza outbreak of the Delmarva Peninsula." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 128 p, 2008. http://proquest.umi.com/pqdweb?did=1597632181&sid=10&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Alejo, Carolina Torres. "Study of the role of quail as reservoirs for avian infectious bronchitis virus." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-29022016-143302/.
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This study aimed to investigate the occurrence and molecular diversity of coronavirus in quail and laying hens raised on the same farms and quail only farms, to determine the role of quail as reservoir for avian infectious bronchitis virus (IBV). To this end, two investigations were carried out, one in the São Paulo state, Southeastern Brazil, in 2013, when some farmers started quail vaccination with Massachusetts IBV serotype after surveillance carried out in 2009-2010 and the other in two regions of Northern Italy, in 2015. In the Brazilian study, samples were collected as pools of tracheas, lungs, reproductive tract, kidneys and enteric contents from quail (Coturnix coturnix Japonica) and laying hens showing IB-like symptoms, while, in the Italian study, samples were collected as pools of tracheal and cloacal swabs and intestine/enteric content from European quail (Coturnix coturnix), showing enteric disorders. All samples were tested by a nested RT-PCR targeted to the 3\'UTR of the Gammacoronavirus genus. Positive samples were submitted to RT-PCR to the RNA-dependent RNA-polymerase gene (RdRp) and two different RT-PCRs to the spike gene, including a typing-multiplex one. Two other RT-PCRs were used to detect the avian metapneumovirus (aMPV) and Newcastle disease virus (NDV). Avian coronavirus was found in all types of samples analyzed in quail and chickens from both type of creations, aMPV subtype B was found in chickens (Brasil) and the NDV was not observed in any samples. Based on the DNA sequences for the RdRp gene, Brazilian and Italian quail strains clustered within either Gammacoronavirus or Deltacoronavirus genus, while, for one Brazilian sample, it was detected co-infection with the two genuses. Phylogeny based on partial S1 subunit sequences showed that the gammacoronaviruses detected in the Brazilian and Italian quail belong to the Brazil type and 793/B, respectively. These results suggest that quail are susceptible to Gamma and Deltacoronavirus and that quail avian coronavirus share spike genes identical to chicken infectious bronchitis virus (IBV); thus, quail might act as reservoirs for avian coronaviruses. Also, Massachusetts vaccination was not efficient to control IBV in Brazilian quail.Este estudo teve como objetivo pesquisar a ocorrência e diversidade molecular de coronavírus em codornas e galinhas criadas nas mesmas propriedades e em codornas criadas em propriedades isoladas, para determinar o papel das codornas como reservatório para o vírus da bronquite infecciosa das galinhas (IBV). Para isso, duas pesquisas foram realizadas, uma em 2013, no estado de São Paulo, Sudeste do Brasil, onde algumas granjas iniciaram a vacinação em codornas contra o IBV com o sorotipo Massachusetts, após um estudo realizado em 2009-2010; e a outra, em 2015, em duas regiões do Norte da Itália. No estudo brasileiro, foram coletados pools de aparelho reprodutor, pulmões, rins, traqueia e conteúdo entérico de codornas (Coturnix coturnix japonica() e galinhas com histórico de manifestações clínicas compatíveis com a Bronquite Infecciosa das galinhas (BIG). Por outro lado, no estudo italiano, as amostras foram coletadas em forma de pools de swabs traqueais e cloacais e intestino/conteúdo entérico de codornas (Coturnix coturnix) com sinais entéricos. Estas amostras foram testadas para os coronavírus aviário (Gammacoronavirus) mediante uma semi-nested RT-PCR dirigida a região não-traduzida 3 (3´UTR). As amostras positivas foram submetidas a RT-PCR do gene codificador da proteína RNA-polimerase RNA-dependente (RdRp) e duas RT-PCRs, incluindo uma multiplex dirigidas a proteína de espícula (S) do vírus da BIG, para genotipagem. Além disso, a detecção de metapneumovírus aviário (aMPV) e o vírus da doença de Newcastle (NDV) também foi realizada por meio de RT-PCRs. Coronavírus aviários foram encontrados em todos os tipos de amostras estudadas em codornas e galinhas de todos os tipos de criações, aMPV subtipo B foi encontrado em galinhas (Brasil) e o NDV não foi encontrado em nenhuma amostras. Com base nas sequências de DNA para o gene codificador da proteína RdRp, as amostras brasileiras e italianas foram agrupadas no gênero Gamma ou Deltacoronavirus, enquanto que, em uma amostra brasileira, foi detectada coinfecção pelos dois gêneros. A filogenia com base nas sequências parciais da subunidade S1da proteína de espícula, evidenciou que os Gammacoronavirus detectados nas codornas brasileiras e italianas pertencem ao genótipo Brasil e 793/B, respectivamente. Estes resultados sugerem que as codornas são suscetíveis aos coronavírus do gênero Gamma e Delta e os coronavírus aviários das codornas compartilham genes de espícula idênticos aos do IBV. Desta forma, sugere-se que as codornas podem servir como reservatórios para coronavírus aviários e que a vacinação com o sorotipo Massachusetts não foi eficiente no controle de IBV nas codornas brasileiras.
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Lopez, Juan Carlos. "The effect of environmental stressors on the immune response to avian infectious bronchitis virus." Lincoln University, 2006. http://hdl.handle.net/10182/643.
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The first aim of this research was to determine the prevalence of IBV in broilers within the Canterbury province, New Zealand, in late winter and to search for associations with management or environmental factors. The second aim was to study how ambient stressors affect the immune system in birds, their adaptive capacity to respond, and the price that they have to pay in order to return to homeostasis. In a case control study, binary logistic regression analyses were used to seek associations between the presence of IBV in broilers and various risk factors that had been linked in other studies to the presence of different avian pathogens: ambient ammonia, oxygen, carbon dioxide, humidity and litter humidity. Pairs of sheds were selected from ten large broiler farms in Canterbury. One shed (case) from each pair contained poultry that had a production or health alteration that suggested the presence of IBV and the other was a control shed. Overall, IBV was detected by RT-PCR in 50% of the farms. In 2 of the 5 positive farms (but none of the control sheds) where IBV was detected there were accompanying clinical signs that suggested infectious bronchitis (IB). Ambient humidity was the only risk factor that showed an association (inverse) with the prevalence of IBV (p = 0.05; OR = 0.92). It was concluded within the constraints of the totally enclosed management systems described, that humidity had an influence on the presence of IBV, but temperature, ammonia, carbon dioxide, oxygen or litter humidity had no effect. In another study environmental temperatures were changed in order to affect the biological function and adaptive capacity of chickens following infection with IBV. The 'affective states' of the animal were assessed by measuring levels of corticosterone (CORT) in plasma and tonic immobility (TI). It was found that low (10 +/- 2°C) and high (30 +/- 2°C) temperatures exacerbated the respiratory signs and lesions in birds infected with IBV as compared to those housed at moderate (20 +/- 2°C) temperatures. The chickens housed at high temperatures showed significantly decreased growth, a higher proportion of hepatic lesions (principally haemorrhages) and a longer tonic immobility period, but there was no significant alteration in the plasma levels of CORT. The birds housed at low temperatures developed a higher proportion of heart lesions (hydropericardium, ventricular hypertrophy) and had significantly higher levels of plasma CORT than birds housed under moderate and/or high temperatures. The specific antibody response to IBV decreased in birds housed under high temperatures. Interestingly the birds housed at high temperatures developed significantly higher levels of haemagglutinin antibodies to sheep red blood cells (SRBC) than those birds housed under low or moderated temperatures. Cell mediated immunity was not significantly affected by heat or cold stress in the first 13 days of treatment but at 20 days the levels of interferon gamma in the birds subjected to low temperatures were lower than in the high temperature group. In other trials, the exogenous administration of low physiological doses of oral CORT (as compared to high pharmacological doses typically used in such experiments) to birds resulted in suppression or enhancement of the immune response depending on duration of treatment and/or dose and nature of the antigen. To our knowledge, this is the first study to show that exogenous CORT can produce an enhancement in the immune response in chickens. iv In conclusion, environmental stressors such as high or low temperatures do affect the physiology of the fast-growing broiler. The adjustments the birds have to make to maintain homeostasis impacts on the course of common infectious diseases, such as IB, that normally is mild in the New Zealand poultry industry. The administration of exogenous CORT showed that this hormone may be part of the physiological stress response and acts as a messenger to prepare the immune system for potential challenges (e.g., infection).
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Kottier, Sanneke Annet. "Investigation into the use of recombination for the production of site-specific mutants of coronavirus infectious bronchitis virus." Thesis, University of Reading, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295313.
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Mockett, A. P. A. "Studies of the humoral immune system of the chicken and its response to avian infectious bronchitis virus infection." Thesis, University of Liverpool, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372722.
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Neuman, Benjamin. "Use of defective-RNAs containing reporter genes to investigate targetted recombination in avian infectious bronchitis virus." Thesis, University of Reading, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367694.
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Sandri, Thaisa Lucas. "Vírus da bronquite infecciosa das galinhas (IBV): distribuição, diversidade molecular e genealogia a partir de amostras de múltiplos órgãos de diversos tipos de criação do plantel avícola brasileiro." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-21122010-105658/.
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A bronquite infecciosa das galinhas (BIG) é uma doença altamente contagiosa causada por múltiplos genotipos/sorotipos do vírus da bronquite infecciosa das galinhas (IBV), um coronavirus do grupo 3. Embora classicamente associado ao trato respiratório, alguns tipos de IBV têm sido descritos com tropismo pelos rins e pelos tratos reprodutivos e entéricos, o IBV pode ser detectado em diversos tipos de tecidos, e também pode acometer aves de todas as idades. Este estudo tem como objetivo verificar a freqüência do IBV em amostras de diversos órgãos e conteúdo entérico de avós, matrizes, poedeiras comerciais e frangos de corte, genotipar as amostras detectadas e estudar a diversidade molecular entre as amostras brasileiras de IBV. Um total de 844 pools de diversos órgãos e conteúdos entéricos provenientes de 200 lotes de avós, matrizes, poedeiras comerciais e frangos de corte, das regiões Sul, Sudeste, Centro-oeste e Nordeste do Brasil, colhidas durante o período de 2007 a 2009 foram testadas para a presença de IBV com um RT-PCR dirigido à região não traduzida 3′(3′UTR). As aves amostradas apresentaram sinais clínicos compatíveis com a BIG. Todas as amostras de IBV detectadas foram tipificadas utilizando uma RT-PCR dirigida ao gene de espícula do vírus. Dezenove amostras tipificadas como variante foram submetidas ao seqüenciamento parcial da região codificadora da subunidade S1 e à análise genealógica. Considerando os pools de órgãos e de conteúdo entérico, 45,50% foram positivos para a presença de IBV, dos quais, 84,63% pertencem ao genotipo Variante e 9,89% ao sorotipo/genotipo Massachusetts. Considerando os lotes, 73,50% foram positivos para IBV, sendo 77,55% variantes e 6,12% Massachusetts. A análise genealógica revelou quatro linhagens virais, todas agrupadas em um exclusivo grupamento de genotipo brasileiro. Estes resultados demonstram que o IBV está disseminado em todas as regiões avícolas brasileiras, com um predomínio massivo de genotipos não Massachusetts e uma elevada diversidade molecular, que deve ser levada em consideração para desenvolver medidas preventivas contra o IBV.Infectious bronchitis (IB) is a highly contagious disease of poultry caused by multiple geno/serotypes of avian infectious bronchitis virus (IBV), a group 3 coronavirus. Though classically associated to the respiratory tract, IBV strains also have been described which harbor tropism for the kidneys and the reproductive and enteric tracts, and might be detected in multiple tissues and can also affect birds of all ages. This survey aimed to assess the frequency of in multiple organs and enteric content samples from grandparents, breeders, layers and broilers, to genotype the IBV strains detected and to study the molecular diversity amongst Brazilian IBV strains. A total of 844 pools of multiple organs and enteric contents from 200 flocks of grandparents, breeders, layers and broilers from the Southern, Southeastern, Central-Western and Northeastern Brazilian regions collected between 2007 and 2009 was screened for the presence of IBV with an RT-PCR target to the 3 untranslated region (UTR). The sampled birds presented symptoms compatible with IB. All IBV strains detected were then typed using an RT-PCR target to the spike gene of the virus. Nineteen strains type as variants were submitted to partial sequencing of the S1 coding region and genealogic analysis. Regarding the organs and enteric content pools, 45.50% were positive for the presence of IBV, from which 84.63% were variant and 9.89% Massachusetts. Taking into account the flocks, 73.50% were positive for IBV, being 77.55% variants and 6.12% Massachusetts. Genealogic analysis revealed four viral lineages, all grouped in an exclusive Brazilian genotype cluster. This results shown that IBV is widespread in all Brazilian poultry regions, with a massive predominance of non-Massachusetts genotypes and a high molecular diversity, which must be taken into account in order to develop preventive measures against IB.
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Luciano, Renato Luís [UNESP]. "Desenvolvimento da técnica de RT-PCR-ELISA para a detecção do vírus da bronquite infecciosa das aves (VBI)." Universidade Estadual Paulista (UNESP), 2003. http://hdl.handle.net/11449/94870.
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Um método de diagnóstico baseado na associação entre as técnicas de RT-PCR e ELISA foi desenvolvido para a detecção do vírus da bronquite infecciosa das aves (VBI). Inicialmente, a especificidade e a sensibilidade analíticas dessa técnica, bem como dos métodos de RT-PCR e Nested-RT-PCR, foram determinadas, tendo-se demonstrado, para o primeiro parâmetro, apenas a detecção do VBI, enquanto que outros vírus heterólogos aviários testados, tais como pneumovírus aviário (APV / estirpe do grupo B), vírus da doença de Newcastle (NDV / estirpe La Sota) e vírus da doença de Gumboro (GDV - estirpe Lukert), não foram detectados. Quanto à avaliação da sensibilidade analítica dos métodos empregados neste estudo foi constatado que o RT-PCR-ELISA demonstrou ser 10 vezes mais sensível do que a RT-PCR, enquanto que a reação de Nested-PCR foi 100 vezes mais sensível que a RT-PCR e 10 vezes mais sensível que o RT-PCR-ELISA. A técnica de RT-PCR-ELISA, juntamente com os outros dois métodos de biologia molecular, foram aplicadas para a detecção das estirpes H-120 e A034 do VBI, em amostras de pulmão e traquéia obtidas de aves experimentalmente infectadas com essas duas estirpes virais. Os resultados obtidos nos diferentes métodos de biologia molecular foram comparados entre si e com a técnica padrão de isolamento viral em ovos embrionados, verificando-se que o RT-PCR-ELISA revelou-se tão específico quanto as técnicas de isolamento viral e Nested-PCR, porém foi menos sensível do que esses mesmos métodos na detecção do VBI. A técnica de RT-PCR-ELISA, utilizada pela primeira vez para o VBI, demonstrou o potencial de se constituir uma alternativa viável para um diagnóstico direto mais rápido e específico do VBI.
A molecular biology method for the detection of avian infectious bronchitis virus (IBV) was developed combining the reverse transcription and polymerase chain reaction (RT-PCR) with the enzyme-linked immunosorbent assay (ELISA). Firstly, the analytical specificity and sensitivity of RT-PCR-ELISA, RT-PCR and Nested-RT-PCR were assessed. The specificity of these methods were confirmed since only IBV was detected, while other heterologous avian viral pathogens such as pneumovirus (Group B), Newcastle disease virus (LaSota strain) and gumboro disease virus (Lukert strain) were not detected by these techniques. The sensitivity of these methods was established, indicating that the RT-PCR-ELISA was about ten times more sensitive than conventional RT-PCR, while Nested-PCR was a hundred more sensitive than RT-PCR and ten times more sensitive than PCR-ELISA. The RT-PCR-ELISA and the two molecular biology methods were also applied for the detection of IBV strains H-120 and A034, in lung and tracheal tissue samples collected from experimentally infected chickens. The results of these different methods were also compared with the standard diagnostic technique, the virus isolation in chicken embryonated eggs, showing that RT-PCR-ELISA was as specific as virus isolation and Nested-PCR, however it was less sensitive than these methods for the IBV detection. The RT-PCR-ELISA, applied for the first time for IBV detection and direct diagnosis in tissue samples, can be potentially applied as an useful alternative for the rapid and specific diagnosis of IBV.
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Toussant, Matthew J. "Studies of egg albumen quality : the effects of dietary vanadium and ascorbic acid, genetics, and avian infectious bronchitis virus vaccination on albumen composition and other relationsips with egg production parameters /." The Ohio State University, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487846885779791.
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Luciano, Renato Luís. "Desenvolvimento da técnica de RT-PCR-ELISA para a detecção do vírus da bronquite infecciosa das aves (VBI) /." Jaboticabal : [s.n.], 2003. http://hdl.handle.net/11449/94870.
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Orientador: Hélio José MontassierBanca: José Moacir Marin
Banca: Liana Brentano
Resumo: Um método de diagnóstico baseado na associação entre as técnicas de RT-PCR e ELISA foi desenvolvido para a detecção do vírus da bronquite infecciosa das aves (VBI). Inicialmente, a especificidade e a sensibilidade analíticas dessa técnica, bem como dos métodos de RT-PCR e Nested-RT-PCR, foram determinadas, tendo-se demonstrado, para o primeiro parâmetro, apenas a detecção do VBI, enquanto que outros vírus heterólogos aviários testados, tais como pneumovírus aviário (APV / estirpe do grupo B), vírus da doença de Newcastle (NDV / estirpe La Sota) e vírus da doença de Gumboro (GDV - estirpe Lukert), não foram detectados. Quanto à avaliação da sensibilidade analítica dos métodos empregados neste estudo foi constatado que o RT-PCR-ELISA demonstrou ser 10 vezes mais sensível do que a RT-PCR, enquanto que a reação de Nested-PCR foi 100 vezes mais sensível que a RT-PCR e 10 vezes mais sensível que o RT-PCR-ELISA. A técnica de RT-PCR-ELISA, juntamente com os outros dois métodos de biologia molecular, foram aplicadas para a detecção das estirpes H-120 e A034 do VBI, em amostras de pulmão e traquéia obtidas de aves experimentalmente infectadas com essas duas estirpes virais. Os resultados obtidos nos diferentes métodos de biologia molecular foram comparados entre si e com a técnica padrão de isolamento viral em ovos embrionados, verificando-se que o RT-PCR-ELISA revelou-se tão específico quanto as técnicas de isolamento viral e Nested-PCR, porém foi menos sensível do que esses mesmos métodos na detecção do VBI. A técnica de RT-PCR-ELISA, utilizada pela primeira vez para o VBI, demonstrou o potencial de se constituir uma alternativa viável para um diagnóstico direto mais rápido e específico do VBI.
Abstract: A molecular biology method for the detection of avian infectious bronchitis virus (IBV) was developed combining the reverse transcription and polymerase chain reaction (RT-PCR) with the enzyme-linked immunosorbent assay (ELISA). Firstly, the analytical specificity and sensitivity of RT-PCR-ELISA, RT-PCR and Nested-RT-PCR were assessed. The specificity of these methods were confirmed since only IBV was detected, while other heterologous avian viral pathogens such as pneumovirus (Group B), Newcastle disease virus (LaSota strain) and gumboro disease virus (Lukert strain) were not detected by these techniques. The sensitivity of these methods was established, indicating that the RT-PCR-ELISA was about ten times more sensitive than conventional RT-PCR, while Nested-PCR was a hundred more sensitive than RT-PCR and ten times more sensitive than PCR-ELISA. The RT-PCR-ELISA and the two molecular biology methods were also applied for the detection of IBV strains H-120 and A034, in lung and tracheal tissue samples collected from experimentally infected chickens. The results of these different methods were also compared with the standard diagnostic technique, the virus isolation in chicken embryonated eggs, showing that RT-PCR-ELISA was as specific as virus isolation and Nested-PCR, however it was less sensitive than these methods for the IBV detection. The RT-PCR-ELISA, applied for the first time for IBV detection and direct diagnosis in tissue samples, can be potentially applied as an useful alternative for the rapid and specific diagnosis of IBV.
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Stephan, Thomas Min [Verfasser], Silke [Akademischer Betreuer] Rautenschlein, Georg [Akademischer Betreuer] Herrler, and Egbert [Akademischer Betreuer] Mundt. "Molecular characterization of the spike protein of an avian infectious bronchitis virus (IBV) with cell culture tropism and identification of the underlying determinants / Thomas Min Stephan ; Silke Rautenschlein, Georg Herrler, Egbert Mundt." Hannover : Stiftung Tierärztliche Hochschule Hannover, 2019. http://d-nb.info/1202774873/34.
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Rossa, Giselle Ayres Razera. "Diversidade molecular dos genes codificadores das proteínas não-estruturais Nsp2 e protease Papaína-like e da proteína estrutural S1 de amostras brasileiras do Coronavírus aviário." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-30032015-142540/.
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Coronavírus, incluindo-se o Coronavírus aviário (ACoV), possuem o maior genoma composto por RNA conhecido entre os vírus. Aproximadamente dois terços desse genoma codificam proteínas não estruturais (Nsps), cujas funções parecem estar associadas à replicação e patogênese viral. Até o momento, esses alvos têm sido pouco explorados quanto a sua diversidade em diferentes linhagens de ACoV. O presente estudo teve como objetivo investigar a diversidade dos genes codificadores das proteínas não estruturais Nsp2 e protease Papaína-like (Plpro), utilizando-se linhagens brasileiras de ACoV. Para tanto, 10 linhagens de ACoV, isoladas em ovos embrionados, foram submetidas à RT-PCR direcionada aos genes codificadores de Plpro e Nsp2, seguindo-se o sequenciamento de DNA e a análise filogenética, juntamente com sequências homólogas obtidas no GenBank. Além disso, realizou-se a genotipagem por meio do sequenciamento parcial do gene codificador da proteína de espícula (região S1). Três das amostras virais obtidas e investigadas no presente trabalho apresentaram padrão de segregação discordante para os genes estudados. O isolado CRG I22 agrupou-se com linhagens virais pertencentes ao genótipo Massachusetts para S1 e com o grupamento de ACoVs brasileiros os genes da Nsp2 e Plpro. O isolado CRG I33 agrupou-se com linhagens virais pertencentes ao genótipo brasileiro para s1 e plpro e de maneira divergente para o gene da Nsp2. Para o isolado CRG I38, não foi obtida a genotipagem por s1, entretanto, similarmente ao observado para o isolado CRG I33, esse isolado agrupo-se com linhagens virais brasileiras para o gene plro e de maneira independente para o gene nsp2. As demais linhagens estudadas resultaram na formação de um grupamento especificamente brasileiro de ACoV, para os três genes estudados. Esses achados sugerem a ocorrência de recombinação nessas amostras discrepantes. Quanto às identidades médias entre as sequencias nucleotídicas analisadas, a região de s1 analisada apresentou as menores identidades (73,75% ±16,78), seguido pelo gene plpro (88,06% ±5,7) e do gene nsp2 (92,28% ±4,37), em acordo com a literatura. Assim sendo, os alvos investigados podem constituir ferramentas úteis na epidemiologia molecular do ACoV e na investigação de linhagens recombinantes do vírus. O presente estudo é o primeiro a investigar a diversidade genética de genes codificadores de proteínas não-estruturais em linhagens brasileiras de ACoV. Os resultados aqui apresentados reforçam a existência de um genótipo brasileiro de ACoV, para os 3 genes estudados. Entretanto, discrepâncias pontuais encontradas no padrão genotípico para s1, nsp2 e nsp3 permitem inferir uma diversidade genética maior do que a conhecida até o momento, possivelmente resultante de eventos de recombinação entre ACoVs brasileiros, ACoVs vacinais e outros ainda desconhecidos. Os resultados obtidos auxiliam na compreensão dos padrões e evolução dos ACoVsCoronaviruses, including Avian coronavirus (ACoV), have the largest known RNA genome. Nearly two thirds of its genome codes for non-structural proteins (Nsps), whose functions appear to be linked to viral replication and pathogenesis. Hitherto these targets have been poorly explored regarding the ACoV lineages diversity. The present study aimed to assess the diversity of non-structural protein 2 (nsp2), papain-like protease (plpro) and spike protein (S1 subunit) coding genes, in Brazilian ACoV strains. To this end, 10 ACoV strains, isolated in embryonated eggs, had its 3rd and 5th passages submitted to RT-PCR targeting nsp2, plpro and s1, followed by DNA sequencing and phylogenetic analysis, herewith homologous sequences obtained from GenBank. Three of the ACoV strains sequenced showed a discordant segregation pattern for target genes. CRG I22 strain clustered with Massachusetts genotipe strains for S1, and with Brazilian cluster for nsp3 and plpro genes. CRG I33 strain, clustered with Brazilian strains for S1 and plpro genes, and was divergent for nsp2 gene. For CRG I38 strain, the S1 sequence was not obtained, however, similarly to what was observed for CRG I33, this strain grouped with the Brazilian lineage for plpro gene and was divergent for nsp2 gene. All the other ACoV here sequenced resulted in a specific Brazilian cluster for the three studied genes. Regarding the mean nucleotide identities measured, s1 gene showed the lowest identity (73.75% ±16.78), followed by plpro gene (88.06% ±5.7) and nsp2 gene (92.28% ±4.37), in accordance with previous reported data. Therefore, the targets of the present study are useful tools for ACoV molecular epidemiology studies and for the survey of recombinant ACoV strains. The presented study is the first one investigating the molecular diversity of non-structural proteins coding genes in Brazilian strains of ACoV. Results achieved herein reinforce the data over the circulation of ACoV Brazilian strains in this country, for the three investigated genes. However, divergences found between S1, nsp2 and plpro genetic patters allow inferring a higher molecular diversity than previously known. It is possible that this divergence is due to recombination events between ACoV from vaccines, Brazilian field strains and others still unknown. These results contribute on the comprehension over genetic patters and evolution of ACoV
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Okino, Cintia Hiromi. "Imunidade celular e humoral o trato respiratório de galinhas desafiadas com o vírus da bronquite infecciosa e efeito de subdosagens da vacina na indução da proteção /." Jaboticabal : [s.n.], 2010. http://hdl.handle.net/11449/104624.
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Orientador: Hélio José MontassierBanca: Adolorata Aparecida Bianco Carvalho
Banca: Clarice Weins Arns
Banca: Geraldo Aleixo da Silva Passos Júnior
Banca: Liana Brentano
Resumo: As respostas imunes inatas e adquiridas, incluindo-se aí tanto as mediadas por fatores humorais como celulares normalmente induzidas após a infecção ou vacinação com o vírus da BI (VBI), são caracterizadas por sua grande complexidade e por aspectos relevantes que ainda são pouco conhecidos, no que tange aos elementos capazes de exercer uma ou mais ações efetoras contra esse patógeno e que culminassem na restrição da replicação viral, seguido de sua eliminação do organismo hospedeiro e também no impedimento de lesões mais severas. Isso posto, foi formulado o presente estudo com o fito principal de fazer a avaliação das respostas imunes humorais e celulares em diferentes intervalos pós-desafio com o VBI de aves previamente vacinadas ou não, realizando-se a mensuração de anticorpos no soro e na lágrima, e a quantificação da expressão de genes relacionados às respostas imunes na superfície traqueal, a fim de correlacionar tais parâmetros com o estado de proteção ao desafio. Os resultados demonstraram que os aumentos significativos nos níveis de anticorpos lacrimais dos isótipos IgG e IgA nas aves previamente vacinadas e também na expressão dos genes relacionados às respostas imunes, sobretudo o CD8, a Granzima A e o IFNg foram correlacionados negativamente com um ou mais parâmetros de alterações patológicas traqueais. Constatou-se também, que a memória das respostas imunes humorais e cito-mediadas conferida por uma única vacinação contra a BI no primeiro dia de idade é dependente da dose vacinal administrada
Abstract: Avian infectious bronchitis virus (IB) is a worldwide infectious disease which causes significant economic losses in poultry industry. The innate and acquired immune responses, including whether there mediated by both cellular and humoral factors that are induced after infection or vaccination with IB virus (IBV) are characterized by their higher complexity and for the relevant aspects that are still poorly known, with respect to the elements able to exercise one or more actions against the pathogen and that culminate in the restriction of its propagation and also on your clearance of the host organism. So, this project was formulated with the main done to make the evaluation of cellular and humoral immune responses at different intervals post-immunization or postchallenge with IBV poultry previously vaccinated or not, performing the measurement of antibodies in serum or tears and quantitation of the expression of genes related to immune responses in tracheal surface, correlating these parameters with the protection against IBV. The results showed that both significative increase of IgG and IgA isotypes in tears of previously vaccinated chickens and the expression levels of genes related to immune responses, especially CD8, Granzyme A and IFN g were negatively correlated with one or more parameters of pathological lesions at trachea. Moreover, we found that the immune memory conferred by vaccination against BI on the first day of age is dependent on vaccine dose administered
Doutor
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Okino, Cintia Hiromi [UNESP]. "Imunidade celular e humoral o trato respiratório de galinhas desafiadas com o vírus da bronquite infecciosa e efeito de subdosagens da vacina na indução da proteção." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/104624.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
As respostas imunes inatas e adquiridas, incluindo-se aí tanto as mediadas por fatores humorais como celulares normalmente induzidas após a infecção ou vacinação com o vírus da BI (VBI), são caracterizadas por sua grande complexidade e por aspectos relevantes que ainda são pouco conhecidos, no que tange aos elementos capazes de exercer uma ou mais ações efetoras contra esse patógeno e que culminassem na restrição da replicação viral, seguido de sua eliminação do organismo hospedeiro e também no impedimento de lesões mais severas. Isso posto, foi formulado o presente estudo com o fito principal de fazer a avaliação das respostas imunes humorais e celulares em diferentes intervalos pós-desafio com o VBI de aves previamente vacinadas ou não, realizando-se a mensuração de anticorpos no soro e na lágrima, e a quantificação da expressão de genes relacionados às respostas imunes na superfície traqueal, a fim de correlacionar tais parâmetros com o estado de proteção ao desafio. Os resultados demonstraram que os aumentos significativos nos níveis de anticorpos lacrimais dos isótipos IgG e IgA nas aves previamente vacinadas e também na expressão dos genes relacionados às respostas imunes, sobretudo o CD8, a Granzima A e o IFNg foram correlacionados negativamente com um ou mais parâmetros de alterações patológicas traqueais. Constatou-se também, que a memória das respostas imunes humorais e cito-mediadas conferida por uma única vacinação contra a BI no primeiro dia de idade é dependente da dose vacinal administrada
Avian infectious bronchitis virus (IB) is a worldwide infectious disease which causes significant economic losses in poultry industry. The innate and acquired immune responses, including whether there mediated by both cellular and humoral factors that are induced after infection or vaccination with IB virus (IBV) are characterized by their higher complexity and for the relevant aspects that are still poorly known, with respect to the elements able to exercise one or more actions against the pathogen and that culminate in the restriction of its propagation and also on your clearance of the host organism. So, this project was formulated with the main done to make the evaluation of cellular and humoral immune responses at different intervals post-immunization or postchallenge with IBV poultry previously vaccinated or not, performing the measurement of antibodies in serum or tears and quantitation of the expression of genes related to immune responses in tracheal surface, correlating these parameters with the protection against IBV. The results showed that both significative increase of IgG and IgA isotypes in tears of previously vaccinated chickens and the expression levels of genes related to immune responses, especially CD8, Granzyme A and IFN g were negatively correlated with one or more parameters of pathological lesions at trachea. Moreover, we found that the immune memory conferred by vaccination against BI on the first day of age is dependent on vaccine dose administered
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Ritterbusch, Giseli Aparecida. "Desenvolvimento e avaliação de uma vacina recombinante contra o vírus da Bronquite Infecciosa das Galinhas carreada por adenovírus defectivo." Universidade Federal de Pelotas, 2015. http://repositorio.ufpel.edu.br:8080/handle/prefix/3267.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
O vírus da bronquite infecciosa das galinhas (VBI) é o agente etiológico da Bronquite Infecciosa (BI), uma enfermidade altamente contagiosa que causa grandes perdas econômicas na avicultura. O VBI é um vírus envelopado, que possui genoma constituído de RNA fita simples, que codifica 4 proteínas estruturais, dentre elas a Nucleoproteína (N), que é produzida em grande quantidade na infecção viral e é reconhecidamente imunogênica. O controle da BI se faz com a imunização das aves através da aplicação de vacinas vivas atenuadas, seguidas de vacinação utilizando antígeno inativado, sendo o sorotipo Massachusetts o único liberado para uso no Brasil. Um dos objetivos do presente trabalho foi realizar um estudo exploratório, afim de conhecer a opinião de diferentes segmentos da avicultura sobre a situação atual da ocorrência de BI nos planteis brasileiros e os custos que ela representa. Diante disso, surge então a necessidade do desenvolvimento de vacinas alternativas e seguras para controle da BI, entre elas a utilização de vacinas vetoriais. Dessa forma, com o objetivo de desenvolver uma vacina efetiva no controle da BI, amostras variantes de VBI foram clonadas em adenovírus humano recombinante e utilizadas para transfectar células HEK293, originando adenovírus recombinantes carreadores do gene N do VBI. Estes vírus foram purificados e utilizados como vacinas recombinantes para imunização de aves SPF. Com base nos dados obtidos, observou-se que apesar das diferentes estratégias de vacinação, a BI ainda é considerada uma doença de alta prevalência que continua causando significativas perdas econômicas na produção avícola de corte e postura no Brasil. Os resultados obtidos demonstraram que a vacina recombinante não induziu uma resposta sorológica detectável pelo teste de Elisa comercial utilizado, bem como não reduziu os escores de lesões nos tecidos das aves vacinadas e desafiadas. Assim, a vacina recombinante carreada por adenovírus defectivo expressando o gene N do VBI foi construída e caracterizada, porém se mostrou ineficaz e não induziu suficiente proteção às aves experimentalmente imunizadas frente ao desafio com VBI.
The infectious bronchitis virus (IBV) is the etiologic agent of Infectious bronchitis (IB), a highly contagious disease that causes great economic losses in the poultry industry. The IBV is an enveloped virus that has RNA single strand genome, encoding four structural proteins, among them Nucleoprotein (N), which is produced abundantly in viral infection and is known immunogenic. The IB control is done by immunization of birds by applying live attenuated vaccine, followed by vaccination using inactivated antigen, wherein the Massachusetts serotype is the only released for use in Brazil. One of the goals of the present work was to conduct an exploratory study in order to know the opinion of different segments of the poultry industry on the current situation of the occurrence of BI in Brazilian squads and the costs that it represents. Therefore, the development of alternative and safe vaccines to BI control is necessary, including the use of vectors. In order to develop an effective vaccine to IB control, samples from IBV field variants were cloned into recombinant human adenovirus and used to transfect HEK293 cells, resulting in recombinant adenovirus carriers of the N gene of the IBV. These recombinant viruses were purified and used as vaccines to immunization of SPF chickens. Based on the obtained data, it was observed that despite the different vaccination strategies, IB is still considered highly prevalent disease that causes significant economic losses in Brazilian poultry industry. The results here obtained showed that the recombinant vaccine does not causes detectable positive serological responses by commercial Elisa test in vaccinated chickens and does not reduce the tissues damage in vaccinated and challenged chickens. Thus, the recombinant vaccine carried by defective adenovirus expressing N gene of IBV was constructed and characterized, but seemed to be ineffective and did not induce sufficient protection to experimentally immunized chickens against IBV challenge.
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Elhafi, Giuma Elaref. "Studies on avian infectious bronchitis including viral persistence." Thesis, University of Liverpool, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406664.
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Jayaram, Jyothi. "Studies on the nucleocapsid protein of infectious bronchitis virus." Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/2243.
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Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid (N) protein may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. In the infected cell, N was the only viral protein that was phosphorylated as shown by 32P-orthophosphate labeling and Western blot analysis and with IBV specific polyclonal chicken antibody. Using pulse-labeling with 32Porthophosphate, the IBV N protein was found to be phosphorylated in the virion, as well as at all times during infection of Vero cells. One-hour pulse-chase analysis followed by immunoprecipitation of IBV N using rabbit anti-IBV N polyclonal antibody showed that the phosphate on the protein did not fall below 70% of the maximum and remained stable. The small but reproducible drop in phosphorylation could modulate the various functions of the N protein in the infected cell. Simultaneous labeling with 32Porthophosphate and 3H-leucine of infected CEK cells indicated a 3.5-fold increase in the ratio of the 32P:3H counts per minute (cpm) on the virion N protein as compared to the 32P:3H cpm ratio of the N protein from lysates at 7 h p.i. The 32P:3H cpm ratio of the N protein from virion from infected-Vero cell lysates was 10.5X more than the 32P:3H cpm ratio of the N protein obtained at 7 h p.i. It has been shown that the N proteins from the measles and rabies viruses form helical nucleocapsid-like structures when expressed in bacteria (Schoehn et al., 2001; Warnes et al., 1995). The ability of E. coli expressed IBV N protein to form helical-nucleocapsid-like structures was investigated using transmission electron microscopy. Full-length, purified histidine-tagged IBV N protein formed nucleocapsid-like structures when expressed in bacteria. Because E. coli -expressed histidine-tagged fragments of the IBV N protein did not form helical nucleocapsid-like structures, the full-length protein is probably required for assembly of these structures. The highly conserved IBV N protein was also used as a diagnostic tool in an ELISA for detecting anti-IBV antibody in chicken serum using a specialized microwave called the BIOWAVE. The BIOWAVE improves the processing time for an ELISA.
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Spencer, Kelly-Anne. "Biophysical characterisation of the infectious bronchitis virus nucleocapsid protein." Thesis, University of Leeds, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436017.
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Hodgson, Theresa Susan. "Open reading frames 3a and 3b of Infectious bronchitis virus." Thesis, University of Bristol, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422552.
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Liu, Ding Xiang. "Translational analysis of the coronavirus infectious bronchitis virus messenger RNAs." Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239182.
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Chamorro, Claudia Carranza. "Genetic diversity of avian coronavirus infectious bronchitis detected from commercial poultry in Brazil." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-04032016-154921/.
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Infectious bronchitis virus (IBV) is the causative agent of an economically important disease of poultry. In Brazil this disease causes respiratory, renal and reproductive problems in birds of all ages, despite constant vaccination with the Massachusetts strain H120. This lack of immunological protection is known to be due the genetic variation in the spike glycoprotein of IBV, which is involved in host cell attachment, neutralization and the induction of protective immunity. Brazilian IBV variants resulting of this genetic variation are present since the 80s and this study aimed to epidemiologicaly analyze and molecularly characterize the existing variants during 2010-2015 and perform a bioinformatics analysis of the available sequences of IBV variants in a 40 year period. Of the 453 samples tested, 61.4% were positive for IBV and 75.9% of them were considered variants and were detected in birds of all ages, distributed in all five Brazilian regions. A fragment of 559-566 bp was obtained from 12 isolates, where BR-I was the predominant variant while only one isolate belonged to the BR-II genotype. Bioinformatics analysis of the sequences of 40 years of Brazilian IBV variants was performed and the ratio of non-synonymous substitutions per non-synonymous site (dn) to synonymous substitutions per synonymous site (ds) dN/dS was calculated. It revealed a predominance of codons with non-synonymous substitutions in the first third of the S1 gene and a dN/dS ratio of 0.6757, indicating that this portion of the gene was under negative selection. Additionally prediction of N-glycosilation sites showed that most of the BR-I variants (from 2003 to early 2014) present an extra site at animoacid position 20, while the newest ones lack this feature.Together these results suggest that IBV Brazilian variants had probably suffered drastic mutations in some points between the years 1983 to 2003 and after achieving an antigenic structure effective enough for invasion and replication in their hosts, the selection processes became silent.O vírus da bronquite infecciosa das galinhas (IBV) é o agente causador de uma doença aviária economicamente importante. No Brasil, esta doença ocasiona problemas respiratórios, renais e reprodutivos em aves de todas as idades, apesar da vacinação constante com a cepa Massachusetts H120. Esta falha na proteção conferida pela vacina é ocasionada por mutações nos nucleotídeos do gene da glicoproteína da espícula, a qual está envolvida no processo de interação comas células do hospedeiro, a neutralização e a indução de imunidade protetora. As variantes brasileiras resultantes dessa mutação genética estão presentes desde os anos 80 e este estudo teve como objetivo analisar epidemiologicamente e caracterizar molecularmente os vírus variantes existentes durante 2010-2015 e realizar uma análise bioinformática das sequências disponíveis no GenBank em um período de 40 anos. Das 453 amostras analisadas, 61,4% foram positivas para IBV e 75,9% delas foram consideradas variantes e foram detectados em aves de todas as idades, distribuídos em todas as 5 regiões do Brasil. Um fragmento de 559-566 pb foi obtido a partir de 12 isolados, onde BR-I foi a variante predominante ao contrario que apenas um isolado pertencia ao genótipo BR-II. Análise bioinformática de 40 anos de variantes do IBV brasileiros revelou uma predominância de codões com as substituições não sinónimos no primeiro terço do gene S1 e uma relação dN / dS de 0,6757, indicando que esta porção do gene estava sob selecção negativa. Além disso a previsão de pontos de de N-glicosilação mostrou que a maioria das amostras variantes BR-I (entre o 2003 e início de 2014) apresentam um ponto adicional na posição 20, enquanto as variantes mais novas não apresentam esse ponto de nglicosilação. Estes resultados sugerem que as variantes brasileiras teriam sofrido mutações provavelmente drásticas em alguns pontos do genoma, entre os anos de 1983 a 2003 e depois de atingir uma estrutura antigênica eficaz o suficiente para a invasão e replicação em seus hospedeiros, o processo de seleção mudou para seleção negativa.
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Batra, A. "The role of the PI3K/AKT signalling pathway during avian infectious bronchitis infection." Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/3004963/.
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Infectious bronchitis is a highly contagious respiratory disease that results in reduced egg production and can be fatal in young birds. It has recently been identified as the most economically detrimental disease to the poultry industry. It is caused by the gammacoronavirus infectious bronchitis virus (IBV), which is endemic in most countries worldwide. All viruses modulate cellular processes to establish themselves within the cell. The cellular PI3K/AKT signalling pathway is often modified by viruses and plays a crucial role in the regulation of many cellular processes. In this project the activation of the PI3K/AKT signalling pathway and downstream processes such as apoptosis, translation and macropinocytosis were investigated during IBV infection. Techniques such as western blotting, immunofluorescence, flow cytometry and protein expression were used to determine the effect of IBV infection on modulation of the signalling pathway, as well as the downstream cellular effects of the modulation. This study demonstrates that IBV requires an active PI3K/AKT pathway for efficient replication, and that infection with IBV induces phosphorylation of AKT in a PI3K-dependent manner in mammalian and avian cells. This activation occurs late during infection in mammalian cells. However, in avian cells activation occurs in a biphasic manner at both an early and late time point during infection. To summarise the findings, a model is presented to describe the role of the PI3K/AKT signalling pathway during IBV infection. This study highlights the importance of the PI3K/AKT signalling pathway during IBV infection and may be applied to other human and livestock coronaviruses for development of therapeutics or novel vaccines.
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Manswr, B. M. "Studies on immunopathogenesis and diagnosis of infectious bronchitis virus in chicken." Thesis, University of Liverpool, 2018. http://livrepository.liverpool.ac.uk/3026921/.
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An RT-PCR assay was developed to amplify and sequence the full S1 gene of classical and variant infectious bronchitis virus (IBVs) enriched in allantoic fluid (AF) or the same AF inoculated onto Flinders Technology Association (FTA) cards. Seven IBV strains (M41, D274, 793B, IS/885/00, IS/1494/06, Q1 and QX) were grown in SPF eggs and RNA was extracted from AF. Full S1 gene amplification was achieved by using two primers and products were sequenced using primers; A, SX3, 1050+ and 1380+ to achieve full S1 gene coverage. Following serial dilutions of AF, it was found that detection limits of the partial assay were higher than those of the full S1 gene. Partial S1 sequences exhibited higher than average nucleotide similarity percentages (79%; 352bp) compared to full S1 sequences (77%; 1,756bp), suggesting that the full S1 protocol has greater strain differentiation efficiency. For IBV detection from AF inoculated FTA cards, four serotypes were incubated and stored for up to 21 days at three temperatures; 4°C, room temperature (24°C) and 40°C. RNA extracted and tested with partial and full S1 protocols. Through partial sequencing, all IBVs were successfully detected at all sampling points and storage temperatures. In contrast, for full S1 sequencing, was not able to amplify the gene beyond 14 days of storage or when stored at 40°C. Full S1 sequencing appears to be suited for detection of IBVs enriched in AF, and has limited application for samples directly embedded onto FTA cards. Following Q1-like infection of specific pathogen free (SPF) or two different breeds of broiler chicks, the live body weight of all three types of chicks were significantly reduced. Also, respiratory clinical signs were found in all types of chick. For the infected SPF chicks, all swabs were RT-PCR positive, with the IBV viral load peaking in the trachea and kidneys at 9 dpi, whereas the proventriculus peaked later at 14 dpi. Significant up-regulation in the expression of several genes (IFNα, TLR3, MDA5, LITAF, IL-1β and IL-6) were seen in the trachea and kidneys at 3, 7 and 9 dpi. In the broiler chicks, the immunopathogenesis of Q1 was cross-compared between fast and slow growing broiler birds. For the fast-growing line (Line-A), swabs from the infected group were RT-PCR positive at all sampling days, whereas the slow growing line (Line-B) were positive until 14 dpi. At 7-9 dpi, higher viral loads were found in the trachea, proventriculus and kidney of fast growers compared to slow growers. Mean IBV ELISA antibody titres in the Line-B were higher than Line-A. Tracheal innate immune response showed IFN-α up-regulation only in Line-A but IFN-β was up- regulated in both lines. For TLR3, an up-regulation was seen in Line-A up to 7 dpi, and for all sampling days in Line-B. MDA5 was up-regulated in Line-A and down-regulated in Line-B at 1 dpi. In the kidneys, for Line-A birds, IFN-α and IFN-β were up-regulated at 1 and 1-3 dpi respectively. There was up-regulation in TLR3 in Line-B throughout the study period but not for Line-A. MDA5 was up-regulated in both lines at 7 and 9 dpi. It appears that the immunopathogenesis of IBV Q1 infection in slow growing (Line-B) chicks was milder in terms of the proinflammatory cytokines levels when compared to the fast growers (Line-A), which could be associated with the genetic differences between these breeds. In an attempt to establish an in vitro infectious model to cross-compare the virulence of IBV live vaccines, eight vaccines and three virulent strains of IBVs were assessed in tracheal organ cultures (TOCs). At 24 and 72 hours post infection (hpi), TOC media and tracheal rings were collected and used for RT-PCR, qRT-PCR, measurement of innate immune responses and examination of total apoptotic cells. Differences in virulence were noted between the strains as certain vaccine strains resulted in cilia destruction comparable with the virulent strain. Average cilia motility readings showed that Mass1 and VirMass reached complete ciliostasis at 96 and 72 hpi respectively, whereas, in the 793B and QX groups complete ciliostasis was reached for all strains by 120 hpi. The qRT-PCR analysis revealed decreased viral presence in the media at 24 and 72 hpi. Differences were found between the total apoptotic cell counts in the tracheal rings among virulent and vaccine strains. Down-regulation in mRNA expression of IFN-α at 24 and 72 hpi occurred in all virulent and vaccine infected TOCs. At 24 hpi, there was up-regulation in IFN-β which was down regulated by 72 hpi in virulent infected TOCs. At 24 and 72 hpi, there was up-regulation in the mRNA expression of TLR3 in all vaccine and variant strains. An up-regulation of MDA5 was seen at 24 hpi in Mass serotype strains, vir793B, 793B1 and QX strains. This study demonstrates successful use of the in vitro TOC model for distinguishing differences in virulence and replication rates among the vaccine IBV strains. Four of the vaccine strains used above were included in an in vivo experiment to validate previous data using a chicken host model. Following inoculation of different vaccine strains in SPF chicks, the immunopathogenesis was evaluated. IBV vaccines (Mass1, Mass2, 793B1 and 793B3) were administered by the oculo-nasal route and chicks were monitored daily for clinical signs. At 1, 3, 5, 7, 9 and 14 dpi, OP and CL swabs were collected for virus detection, and blood was collected for antibody assay. Necropsy was carried out on 1, 3, 5, 7, 9 and 14 dpi, with gross lesions observed and tracheal tissues collected for qRT-PCR, immunohistochemistry (CD4+ and CD8+), histopathology and host innate immune gene expressions. Respiratory signs started from 3 dpi. Viral load was lower in the 793B3 group at 5-7 dpi, compared to other groups. Higher expression of IFN-β was seen at 3 dpi in 793B groups, whereas793B1 showed a lower expression of TLR3 at 5-7 dpi compared to other groups. Down-regulation of IL-6 was seen at 7-9 dpi in all inoculated groups except for Mass2. There was higher up-regulation of MYD88 in the tracheal tissue in all inoculated groups. There was higher up-regulation of IFN-γ at 7-9 dpi in Mass1 and Mass2 compared to the control and 793B1 and 793B3. From lachrymal fluid, tracheal washes and tracheal tissue there was higher up-regulation in IgG expression in the all inoculated groups at 9-14 dpi. In Mass2 and 793B3, there was higher up-regulation of IgA in the tracheal tissue at 7-9 dpi. The 793B1 inoculated groups demonstrated higher cell mediated immune responses, represented by higher CD8β gene expression in the period of 5-9 dpi, and higher cell counts of CD4+ and CD8+ at 14 dpi. Results demonstrated that the in vivo were generally comparable to in vitro findings. This demonstrates that large number of IBV live vaccines could be screened for virulence and early immune responses using TOCs, instead of birds.
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Harrison, Sally M. "Characterisation of the interaction of infectious bronchitis virus with cyclin Dl." Thesis, University of Leeds, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485777.
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The cell cycle describes the growth and division of cells and is split into five distinct phases, GO, Gl, S, G2 and M. Each stage of the cell cycle is controlled by both positive and negative regulatory molecules, including cyclins, their partner molecules the cyclin dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CDKIs). The relationship between the cell cycle and virus infection is well characterised for DNA and retroviruses, but less so with RNA viruses. This study 'investigated the relationship between the coronavirus infectious bronchitis viI1}s (mV), (a major pathogen of chickens causing severe economic losses to poultry' producing countries) and the G1 phase cell cycle regulatory molecule cyclin D1. Cyclin D1 has been shown to be reduced in mv-infected cells with an arrest ofinfected cells in the G2 phase of the cell cycle. This study shows that the reduction of cyclin D I is post transcriptional and independent of the cell cycle stage at the 'time of infection. siRNA analysis was also used to investigate' whether the reduction' of cyclin D1 was essential for virus replication. The mechanism of cyclin D1 nuclear export and degradation in mv-infected cells was determined by the use of MG132, which inhibits the cellular proteasome, LiCI, an inhibitor of glycogen synthase three beta (GSK 3~) and leptomycin B (LMB), which inhibits chromosome region maintenance-l (CRM-l) mediated nuclear export. Confocal microscopy and Western blot analysis indicated that LiCI, LMB and MG132 stabilised cyclin D1 levels in mvinfected cells, although a population of cyclin 'D I appeared to be reduced independently of GSK 3~, potentially by a virus mediated mechanism. These studies 'also lead to LiCI possibly being a novel inhibitor ofmv in cell culture. , The virus protein(s) responsible for inducing the phenotypic G2 phase arrest and reduction of cyclin Dr in mY-infected cells was also investigated, using the mv nucleocapsid (N) and accessory protein 5a as candidates. Neither of these proteins however appeared to induce the G2 phentoype, although the mv 5aprotein did appear to reduce cyclin D1. Over expression studies showed that mv 5a induced some cell cycle perturbations, which were then confirmed by the use of reverse genetics in which 'the 5a ORF had been knocked out. Thisleads.to t?e hypothesis that the induction of the G2 phenotype could be regulated by multiple virus proteins.
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Licata, Matthew J. "The efficacy of combined infectious bronchitis/Newcastle disease vaccines." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 112 p, 2007. http://proquest.umi.com/pqdlink?did=1253510101&Fmt=7&clientId=79356&RQT=309&VName=PQD.
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Raj, G. Dhinakar. "Studies on pathogenicity and immunopathogenesis of infectious bronchitis virus infection in chickens." Thesis, University of Liverpool, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321164.
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Hall, Ross Howden. "The role of infectious bronchitis virus accessory proteins 3a, 3b and 4b." Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3028614/.
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Infectious bronchitis (IB) is a respiratory disease in domestic fowl caused by the gammacoronavirus infectious bronchitis virus (IBV). Current approaches to combat this disease are hindered due to vaccines unable to cross-protect against the many different strains of IBV. To develop novel therapies or more crossprotective vaccines, a better understanding of IBV molecular biology is required. IBV is known to express four accessory proteins, 3a, 3b, 5a, and 5b, as well as a sub-genomic RNA that has the potential to code for an additional 11 kDa protein, referred to as 4b. The role of this sub-genomic RNA is not known, as is whether this transcript is translated during infection. IBV accessory proteins are dispensable for replication and are thought to play a role in virulence or pathogenicity. The functions of 3a and 3b are unknown, although they have been shown to play a part in the interferon response in a yet unknown manner. Using in vitro assays and mass spectrometry, the mechanism of action of 3a on the interferon response was determined. IBV 3a inhibits and stimulates interferon expression in a dose-dependent manner by regulating the turnover of interferon signalling proteins, MAVS and IRF7. Flow cytometry has identified a role for IBV 3b in inducing apoptosis during infection, possibly by interacting with apoptotic proteins VDAC2 or BAG6. Lastly, using an antibody raised against the predicted 4b peptide sequence, 4b was detected during infection, confirming it as the fifth IBV accessory protein. Furthermore, mass spectrometry was utilised to identify a role for 4b in regulating cellular translation and the stress granule response. Accessory proteins are highly conserved in the many different strains of IBV and are usually pathogenicity factors making them potential targets for novel therapies. Researching the role of these accessory proteins is essential to understand how IBV causes IB and for the development of more targeted therapies or more cross-protective vaccines.
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Awad, Faez. "Studies on the immunopathogenesis, diagnosis and control of infectious bronchitis and avian metapneumoviruses in chicken." Thesis, University of Liverpool, 2014. http://livrepository.liverpool.ac.uk/2006220/.
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This thesis describes field and experimental investigations on various aspects of the immunopathogenesis, diagnosis and vaccination of infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) in the chicken. The immunopathogenesis of an economically important variant IBV (IS/885/00 like) seen in the Middle East and North Africa was examined in one day old specific pathogen free (SPF) and commercial broiler chicks (Chapter 3). The virus caused respiratory distress, depression and diarrhoea in both SPF and broiler chicks but the severity was milder in the latter. Mild head swelling was observed in one infected broiler chick at 15 days post infection (dpi) and virus with 100% nucleotide level similarity to the inoculum was detected by reverse transcription polymerase chain reaction (RT-PCR) and virus isolation (VI). In the IS/885/00-like infected SPF chicks, cystic oviducts were found in two female chicks. IBV with 99% part S1 sequence similarity to the initial inoculum was isolated from the cystic fluid. The protection provided by current commercial vaccines against variant IBV IS/885/00 like and IS/1494/00 like was investigated in day old commercial broiler chicks (Chapter 4). Protection was evaluated based on the clinical signs, gross lesions, tracheal ciliary scores and virus detection by RT PCR. It was found that administering combined live H120 and CR88 vaccines simultaneously at day old, followed by CR88 vaccine at 14 days old gave more than 80% ciliary protection against both of the Middle East isolates. Cellular and local immune responses in the trachea following vaccination of day old broiler chicks with different live IBV vaccines were evaluated (Chapter 5). In addition, protection conferred against virulent IBV was also examined. All vaccination programmes were able to induce measurable levels of CD4+, CD8+ and IgA bearing B cells in the trachea following vaccination when compared to unvaccinated birds. Expression levels of CD4+ and CD8+ cells varied between the vaccinated groups. Vaccines containing Mass2 combined with 793B2 produced good protection against challenge with virulent IBV QX compared to vaccines containing Mass (Mass1 or Mass2) alone or Mass1 with D274 or CR88. All vaccination programmes produced more than 80% protection against homologous (M41 and 793B) challenge. In Chapter 6, IBVs with high nucleotide level similarity to IS/885/00 like and IS/1494/06 like strains were detected by RT PCR in a broiler flock exhibiting high mortality and respiratory distress in Libya. For the first time, these findings have highlighted the circulation of variant IBVs in the Eastern part of Libya. Humoral and cellular immune responses and protection studies in SPF chicks that received live Newcastle disease virus (NDV), aMPV and IBV vaccines in single, dual or triple combinations were examined (Chapter 7). Protection against virulent IBV or aMPV was not affected when the vaccines were given either singly or in combination. There were no significant differences in the mean antibody titres of the NDV-vaccinated groups and they remained above the protective titre. The mean titres of antibodies against aMPV were suppressed when aMPV vaccine was given with other live vaccines but the aMPV-vaccinated groups were fully protected when challenged with virulent aMPV. The mean titres of antibodies were similar in the IBV-vaccinated groups and all IBV-vaccinated groups gave almost 100% protection against M41 challenge. Between the vaccinated groups, there were no significant differences in the mean numbers of CD4+, CD8+ and IgA-bearing B cells, reflecting similar levels of tracheal cellular and IgA responses irrespective of single, dual or triple vaccine applications. Despite the aMPV humoral antibody suppression, the efficacy of the live vaccines was not compromised when they were given simultaneously to young SPF chicks. Comparative studies in day old SPF chicks using both aMPV subtype A or B, separately or in combination, were evaluated (Chapter 8). There were significant differences in the degree of the clinical signs induced by the single subtypes A, B or A+B given together, with most severe signs observed in the latter two groups. By RT-PCR or VI, subtype B virus persisted longer than subtype A. Even though similar titres of the viruses were used, birds given subtype B alone or in combination showed a greater increase in antibody titres than those given A. These findings demonstrate that for the two strains used, subtype B was more pathogenic than subtype A and was excreted and persisted in the tissues for longer. The use of Flinders Technology Associates (FTA) cards for detection of several avian pathogens has been previously reported. To date, no information has been published on the use of FTA cards for detection of aMPV. In Chapter 9, the feasibility of using FTA cards for the molecular detection of aMPV subtype A and B by RT-PCR was investigated. Findings showed that FTA cards are suitable for collecting and transporting aMPV-positive samples, providing a reliable and hazard-free source of RNA for molecular characterization.
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Saraiva, Giuliana Loreto. "Epidemiologia molecular, filogenia e filogeografia do Infectious bronchitis virus em um panorama global." Universidade Federal de Viçosa, 2015. http://www.locus.ufv.br/handle/123456789/7510.
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A criação de aves em sistemas industrializados vem adquirindo, mundialmente, características que envolvem uma elevada densidade animal, o que, consequentemente, leva ao surgimento de condições epidemiológicas capazes de predisporem ao aparecimento de problemas sanitários. Diante deste contexto, aumenta-se a preocupação com as doenças infecciosas, dentre elas, a Bronquite Infecciosa das Galinhas (BIG), cujo agente etiológico é o Infectious bronchitis virus (IBV). O presente estudo teve como objetivo reconstruir a epidemiologia molecular do IBV, por meio de análises filogenéticas e filogeográficas dos genes estruturais N e S1, para estudo da evolução viral. Estas informações são importantes para estabelecer um padrão de dispersão em um contexto histórico geográfico, entender eventos biológicos no passado relacionados à dinâmica populacional e ajudar no desenvolvimento de estratégias efetivas de controle do IBV. Para isso, foram montados dois bancos de dados com sequências moleculares completas e parciais dos genes N e S1 obtidos no GenBank. O primeiro banco de dados (IBV1) contemplou sequências completas de S1 (N=256) e N (N=154) e o segundo banco de dados (IBV2) foi montado com sequências parciais de S1 (N=390) e N (N=173). Importantes parâmetros para estudo da evolução viral foram analisados nesse trabalho para os genes S1 e N do IBV, incluindo estimativas de eventos de recombinação, taxa de mutação, pressão de seleção, dinâmica populacional e dispersão espacial. Os resultados apontam que, na ausência de uma elevada taxa de mutação e pelo fato da seleção purificadora ser a principal forma de seleção natural no IBV, o processo de recombinação tem um papel fundamental na evolução do IBV. As análises de dinâmica populacional sugerem que, até meados da década de 2000, os diferentes programas de vacinação, juntamente com os métodos de biosseguridade, foram eficazes para controlar o crescimento do tamanho efetivo populacional do IBV. Contudo, a partir de 2007, o aumento da população de variantes do IBV, pode indicar que os protocolos vacinais, usados no presente, não estão sendo mais eficazes. Os resultados das análises de dispersão viral do gene S1 e N foram analisados de forma conjunta, a fim de reconstruir um padrão de dispersão do IBV ao longo de sua história evolutiva. O ponto de origem de todas as cepas atuais do IBV foi a China que, após dispersão local, disseminou para os Estados Unidos e, por conseguinte, se dispersou para diferentes países e continentes até alcançar a ampla distribuição mundial encontrada nos dias atuais. Os principais centros de dispersão atuais do IBV foram a China, Estados Unidos, Brasil, Taiwan e Europa.
The intensification of poultry in industrial systems has acquired, worldwide, features that involve a high stock density, which, consequently, leads to the emergence of epidemiological conditions that may predispose to the appearance of health problems. Given this context, the concern with infectious diseases increases, among the diseases is the Avian Infectious Bronchitis (IB), whose etiologic agent is the Infectious bronchitis virus (IBV). This study aimed to reconstruct the molecular epidemiology of IBV through phylogenetic and phylogeographic analyzes of structural genes N and S1, to the study of viral evolution. These pieces of information are important to establish a pattern of dispersion in a geographical historical context, to understand biological events in the past related to population dynamics and to help develop effective strategies for control of IBV. To establish a better outlook for discussion of IBV molecular epidemiology, there were created two databases with complete and partial molecular sequences of genes N and S1 obtained from GenBank. The first database (IBV1) considered complete sequences of the S1 (N = 256) subunit and of the N (N = 154) gene of IBV. To increase the extension of countries from which sequences are available, a second database (IBV2) was created with partial sequences of the S1 (N = 390) gene and the N (N = 173) gene. Important parameters for the study of viral evolution were analyzed in this work for the S1 and N genes of the IBV, including estimates of recombination events, mutation rate, selection pressure, population dynamics and spatial dispersion. The results show that, in the absence of a high mutation rate and because of the purifying selection to be the main form of natural selection in IBV, the recombination process plays a key role in the evolution of IBV. The analysis of population dynamics suggest that, until the mid-2000s, the different vaccination programs, along with the biosecurity methods have been effective in controlling the growth of effective population size of the IBV. However, since 2007, the increase in the population of IBV variants may indicate that the vaccine protocols, used in the present, have not been effective anymore. The results of analyzes of the viral spread of the gene S1 and N were analyzed together, in order to reconstruct a pattern of the IBV dispersion along its evolutionary history. The point of origin of all current strains of IBV was China, which after local dispersion spread to the United States of America and thus dispersed to different countries and continents until reaching the worldwide distribution found today. The current main scattering centers of the IBV were China, the United States, Brazil, Taiwan and Europe.
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Dashan, Li. "Factors affecting the membrane fusion-inducing capacity of the spike protein of avian infectious bronchitis coronavirus (IBV)." Thesis, Royal Veterinary College (University of London), 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522192.
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Stirrups, Kathleen Elizabeth. "A defective-RNA expression vector for targeted recombination of the coronavirus infectious bronchitis virus." Thesis, University of Reading, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285965.
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Knoetze, Adrian David. "Investigation into the variation of infectious bronchitis virus serotypes in KwaZulu-Natal poultry flocks." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/40700.
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Infectious bronchitis virus (IBV) is a member of family Coronaviridae and is classified into group 3 of the Coronaviruses. The virus is a single-stranded positive-sense RNA virus with a genome of 27kbp. IBV is a highly infectious disease of chickens that results in high morbidity with moderate to severe mortality depending on the strain involved, age of the birds, and immune status of the chickens. Multiple worldwide investigations indicate that differentiation within the S1 glycoprotein gene can lead to serotype variation within the IBV species. In this study 46 isolates collected over two years from broiler and broiler breeder flocks and eight historical isolates were analyzed. Forty one isolates originated from the KwaZulu-Natal region whilst the remaining thirteen were isolated from 4 other poultry-dense provinces. The S1 gene was sequenced and compared to determine variation between South African isolates, as well as global sequences submitted to Genbank. The results indicate the division of isolates analyzed into 2 different clades of IBV within the province. The most prevalent genotype was similar to IBV Mass strain detected in 79% of the full S1 sequences. Variation up to 22.3% was detected within local strains, supporting the hypothesis that multiple IBV serotypes may co-circulate in the same region simultaneously. Additionally, more conservation was observed among Mass serotypes versus QX-like serotypes, implying that vaccine use can influence the variability within the IBV population. Higher variability was found in the first half of the S1 gene in comparison to the last half of the S1 gene. This is in agreement with previous findings that the hypervariable regions of the S1 gene are located within the first 450 base pairs. This study offers the first published consolidation of IBV isolates from South Africa and identifies variation within the IBV population of the SA broiler flock. Previous publications list four or five IBV isolates whilst this study describes variation found in 54 isolates spanning 32 years. In addition this study provides the insight into the prevalence of IBV variation in poultry flocks due to the large number of isolates. The comparative use of geno- and serotyping for South African IBV isolates is also described for the first time in this study.Dissertation (MSc)--University of Pretoria, 2013.
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Veterinary Tropical Diseases
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Youn, Soonjeon. "In vitro assembly of an infectious cDNA clone of infectious bronchitis virus and its application as a gene transfer vector." Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/1311.
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An infectious cDNA clone of Vero cell adapted Beaudette strain of IBV was constructed using in vitro assembly of cDNA fragments. The entire genome of IBV was RT-PCR amplified into seven fragments, with each piece overlapping about 10 nucleotides. The fragments were ligated and transcribed to synthesize RNA, which was transfected into BHK-21 cells. These cells were then overlaid onto IBV susceptible Vero cells. After five days transfection, the virus was successfully rescued from the transfected cells. The cDNA clone from our laboratory strain has a five nucleotide insertion not present in the originally sequenced virus, resulting in total genome size of 27,613 nucleotides. The infectious cDNA clone was further manipulated to demonstrate its potential as a gene transfer vector, by replacing the ORF5a open reading frame with enhanced green fluorescent protein. The recombinant infectious cDNA clone was also successfully rescued after three days transfection of BHK-21 cells followed by co-culturing with Vero cells. This study showed that the 5a protein, whose function is not known, is not necessary for in vitro IBV replication. This study also showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.
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Mondal, Shankar Prosad. "Molecular characterization of the features of antigenic and pathogenic variant strains of infectious bronchitis virus /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2005. http://uclibs.org/PID/11984.
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Bickerton, Erica Jane. "Cellular tropism and cell-to-cell fusion properties of the infectious bronchitis virus spike glycoprotein." Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/35165/.
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There are numerous vaccines available for the control of infectious bronchitis virus (IBV) in poultry, however protection is short-lived and poorly cross-protective between strains. The vaccines must currently be grown in embryonated eggs, a cumbersome and expensive process. The ability to grow vaccines on a cell-line such as Vero cells would be highly advantageous. The spike (S) glycoprotein of IBV is comprised of two subunits, S1 and S2, has a vital role in virulence in vivo and is responsible for cellular tropism in vitro. This project aims to identify the amino acids present in the S glycoprotein involved in determination of cellular tropism and cell-to-cell fusion. The IBV Beaudette strain is able to replicate in both primary chick kidney (CK) cells and Vero cells, whereas the IBV M41 strain replicates in primary cells only. Recombinant IBVs with chimaeric S genes were generated using a reverse genetics system with the genomic background of Beaudette and part of the S gene from M41. Their growth characteristics and cellular tropism were investigated. The S2 subunit of Beaudette was found to be sufficient to confer the ability to grow on Vero cells and swapping just three amino acids with corresponding ones from M41 was sufficient to remove the ability of the Beaudette S glycoprotein for growth on Vero cells. Beaudette was further adapted to syncytia formation on Vero cells by serial passage and isolates were sequenced to identify amino acid changes between parent and Vero-adapted viruses that are potentially involved in cell-to-cell fusion. Understanding the way in which IBV infects host cells is vital in order to rationally design better vaccination and treatment strategies and help to reduce the prevalence of IBV infection in poultry worldwide. Using the IBV reverse genetics system, we now have the potential to grow IBV vaccines on Vero cells.
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Penzes, Zoltan. "Defective replicating RNAs of coronavirus infectious bronchitis virus : investigation of replication and genome packaging signals." Thesis, University of Hertfordshire, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283879.
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Pereira, Claiton Gonçalves. "Caracterização biológica e molecular de estirpes vacinais e isolados de campo de Infectious bronchitis virus." Universidade Federal de Viçosa, 2015. http://www.locus.ufv.br/handle/123456789/7565.
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Infectious bronchitis virus (IBV) é uma das principais causas de perdas econômicas na indústria avícola, afetando galinhas de todas as idades, apesar das tentativas de controle por vacinação. Os objetivos deste estudo de três capítulos propôs-se: 1) analisar a composição genética das vacinas vivas H120 e Ma5 de IBV disponíveis comercialmente no Brasil, 2) promover a caracterização molecular de IBV acometendo um plantel de matrizes e, 3) investigar persistência viral nesse lote ao final do ciclo de produção e verificar a ocorrência de transmissão vertical. No capítulo 1 foi obtida a sequência completa do gene S1 das vacinas produzidas por diferentes laboratórios, sendo verificada que as vacinas H120 possuem subpopulações de IBV distintas, refletindo em até 14 diferenças na sequência de aminoácidos entre as vacinas. Na análise do cromatograma de S1 obtida da vacina Ma5 também foi detectado pico secundário, sugerindo a presença de subpopulação do vírus. Para abordar essa questão foi desenvolvido um sistema para identificar a população de espécies de RNA dessa vacina. Apesar de ser clonada foram identificadas pelo menos cinco subpopulações do vírus intra-vacina. Portanto, essa grande diversidade genética entre as vacinas poderia justificar os diferentes resultados da vacinação. No capítulo 2, a sequência parcial de S1 de IBV foi obtida diretamente de amostras clínicas. A vacina Ma5, previamente utilizada nesse lote, foi detectada na traqueia e no intestino delgado; enquanto subpopulação de IBV variante foi identificada em órgão específico das aves. Esse achado tem grande importância sob o ponto de vista aplicado porque ele ilustra o potencial repertório patogênico que a infecção por IBV de campo pode imputar sobre um lote de galinhas acometidas pela doença. Finalmente, no capítulo 3, diferentes órgãos dessas galinhas em final de produção e líquido cório-alantoide (LCA) de embriões vivos com 18 dias de incubação oriundos desse lote foram utilizados para a detecção e identificação de IBV. A vacina Ma5 foi detectada no intestino delgado, rins e oviduto das galinhas e no LCA de embriões. No entanto, nas tonsilas cecais foi detectado IBV com significativa diferença genética na comparação com a vacina Ma5 previamente utilizada e aqueles identificados nos diferentes órgãos das aves na época da doença. Esses achados sugerem que além da vacina, outras subpopulações de IBV podem persistir por longos períodos no hospedeiro e, pelo menos em galinhas em final de produção a vacina Ma5 pode ser transmitida verticalmente.
Infectious bronchitis virus (IBV) is a major cause of economic losses in the poultry industry, affecting chickens of all ages, despite attempts to control by vaccination. The objectives of this three-chapter study were: 1) analyze the genetic makeup of the 120 and Ma5 IBV vaccines strains available commercially in Brazil, 2) promote the molecular characterization of IBV affecting one broiler breeding flock and, 3) to investigate IBV persistence in this flock at the end of the production cycle and to verify the occurrence of vertical transmission. In chapter 1, the complete sequence of the S1 gene of vaccines produced by different laboratories was obtained and verified that the vaccines of the strain H120 have different subpopulations, resulting in up to 14 amino acid sequence substitutions among the vaccines. On the other hand, the analysis of Ma5 strain S1 gene an additional peak was detected suggesting the presence of subpopulation of the virus. To address this issue a system to identify the population of RNA species of the vaccine was developed. Despite being cloned, at least five subpopulations of genotypes were detected with in a Ma5 batch. Therefore, this great genetic diversity among the vaccines could justify the different vaccination reactions/results. In chapter 2, partial sequence of S1 of the IBV was obtained directly from clinical specimens. In alignment with the Ma5, which was previously used therein, the same was detected in the trachea and small intestine; while subpopulation of IBV variant was identified in specific organ of birds. This finding has great importance from the applied point of view because illustrates the potential pathogenic repertoire that infection by field IBV can charge on the same lot of chickens affected by the disease. Finally, in chapter 3, different organs of breeders at the end of production and allantoic fluid of embryos with 18 days of incubation deriving this flock were used for the detection and identification of IBV. The Ma5 vaccine was detected in small intestine, kidneys and oviduct of the chickens, and also in the allantoic fluid embryos. However, in the cecal tonsils, IBV was detected in significant genetic difference in comparison with the previously used Ma5 vaccine and those detected in different organs of the birds at the time of disease. These results suggest that in addition to vaccine, other (s) subpopulation (s) of IBV can persist for long periods in the lost least chickens at the end of production Ma5 vaccine can be transmitted vertically.
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Martini, Matheus Cavalheiro 1983. "Estudo experimental em camundongos e aves comerciais com isolado de pombo do vírus da bronquite infecciosa (IBV) = Experimental study in mice and poultry with isolated from pigeon infectious bronchitis virus (IBV)." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316633.
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Orientadores: Clarice Weis Arns, Helena Lage FerreiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O vírus da Bronquite Infecciosa (VBI), pertencente à família Coronaviridae, é um importante patógeno à sanidade e fatores econômicos da produção avícola no Brasil e no mundo. O VBI possui múltiplos sorotipos e o frequente surgimento de novas variantes é um dos principais problemas relacionados a este vírus. Este trabalho tem como objetivo a investigação experimental da patogênese de um isolado de pombo (Columba/Brazil/2007/Unicamp/67T), caracterizado molecularmente pelo gene S1 como VBI sorotipo Massachusetts, e seus efeitos in vivo, em galinhas e camundongos. O presente estudo foi dividido em duas partes, na primeira um grupo de aves "specific pathogen free" (SPF) foi inoculado pela via óculo-nasal com a amostra viral proveniente de pombo. Os animais, de um dia de vida, foram sacrificados nos dias 2, 4, 5, 7, 9, 11, 14, 21, 28, 35 e 42 dias pós-inoculação (dpi). Foram coletados suabes de traqueia, seio nasal e cloaca, além de órgãos como pulmão, íleo, pró-ventrículo (coletado entre 7 e 21 dpi), rim, tonsilas cecais (coletada a partir de 4dpi) e testículos (coletado a partir de 5 dpi). Sinais clínicos respiratórios como espirros, estertores, corrimento nasal, além de letargia, diarreia e perda de coordenação foram observados principalmente no 5dpi. A inibição da atividade ciliar ocorreu concomitantemente ao pico de sinais clínicos das aves. Foi analisado tropismo tecidual, através da quantidade de RNA viral detectado, pelo trato digestório. Os maiores títulos de RNA viral foram detectados na tonsila cecal, seguida pelo íleo (ambos no 5dpi) e cloaca (no 2dpi). Além disso, houve detecção de RNA viral no rim e trato respiratório, com maior título de RNA viral na traqueia. Os órgãos que apresentaram maiores danos teciduais através do exame histopatológico foram o rim, íleo e traqueia (todos no 5dpi). Por fim, as aves inoculadas com a amostra do VBI oriundo de pombo produziram anticorpos entre os dias 14 e 21dpi, detectados no soro destes animais através do ELISA. Na segunda parte do trabalho, a capacidade de replicação de diferentes variantes do VBI em camundongos foi avaliada. Para tanto, camundongos das linhagens Balb/C e A/J foram inoculados pela via nasal com duas amostras do sorotipo Massachussets (Mass) e com a variante brasileira (BR-I), e sacrificados no 3, 10 e 14 dpi. Não foram observados sinais clínicos nem lesões macroscópicas graves. O RNA viral foi detectado em todos os órgãos coletados, sendo os principais órgãos de replicação o seio nasal e pulmão (no 3dpi) para os camundongos da linhagem A/J e pulmão e duodeno (ambos no 3dpi) na linhagem de camundongos Balb/C, nos quais os títulos virais detectados foram mais altos. Pneumonia intersticial, edema e infiltrado mononuclear foram as principais alterações histopatológicas observadas no 3dpi em camundongos inoculados com as diferentes variantes. A presença da nucleoproteína viral, pela imunohistoquímica, foi detectada no duodeno, traqueia e pulmão de camundongos no 3dpi nas duas linhagens de camundongos. Os anticorpos contra o coronavírus aviário foram detectados somente no 3dpi. Assim, os resultados do presente estudo demonstraram que a variante Massachussets, com origem de pombo, causa a doença clínica em aves comerciais não vacinadas e pode replicar em modelo mamífero por um curto período de tempo, ressaltando a importância da vacinação e o papel potencial dos roedores como possível reservatório e carreador do vírus
Abstract: Infectious bronchitis virus (IBV) belonging to the family Coronaviridae is an important pathogen to sanity and economics of poultry production in Brazil and worldwide. The VBI has multiple serotypes and the frequent emergence of new variants is one of the main problems related to this virus. This work aims to experimentally investigate the pathogenesis of pigeon sample (Columba/Brazil/2007/Unicamp/67T), molecularly characterized by S1 gene as IBV Massachusetts serotype, and its effects in vivo in chickens and mice. This study was divided into two parts. In the first part, a group of birds "specific pathogen free" (SPF) was inoculated by oculo-nasal route with the viral sample from pigeon. The animals with one-day-old, were sacrificed on 2, 4, 5, 7, 9, 11, 14, 21, 28, 35 and 42 days post-inoculation (dpi). Tracheal swabs, nasal sinus and cloaca were collected, and organs such as lung, ileum, pro-ventricular (collected between 7 and 21dpi), kidney, caecal tonsils (collected from 4dpi) and testes (collected from 5 dpi). Clinical signs such as sneezing, rales, nasal discharge, lethargy, diarrhea, and loss of coordination were observed mainly in the 5dpi. Inhibition of ciliary activity occurred concomitantly with the peak of clinical signs of birds. Tissue tropism was analyzed by the amount of viral RNA detected by the gastrointestinal tract. The higher titers of viral RNA were detected in the cecal tonsil, followed by the ileum (both in 5dpi) and cloaca (in 2dpi). In addition, viral RNA was detected in the kidney and respiratory tract, with highest titer of viral RNA in the trachea. The organs that showed severe tissue damage by histopathology were the kidney, ileum and trachea (all in 5dpi). Finally, the birds inoculated with the sample originated from IBV Pigeon produced antibodies between 14 and 21dpi, detected in the serum of these animals by ELISA. In the second part, the replication capacity of different variants of IBV in mice was evaluated. For this, mice of strains BALB/C and A/J were inoculated intranasally with two strains of Massachusetts (Mass) serotype and the Brazilian variant (BR-I), and sacrificed at 3, 10 and 14 dpi. No clinical signs or severe macroscopic lesions were observed. The viral RNA was detected in all organs collected, higher tittles were detected on sinus and lung (in 3dpi) for mice of strain A/J and on lung and duodenum (both in 3dpi) in the line of Balb/C; in this line the viral titles were higher than the strain A/J. Interstitial pneumonia, edema and mononuclear cell infiltration were the main histopathological changes observed in 3dpi in inoculated mice with different variants. The presence of viral nucleoprotein, immunohistochemistry was detected in the duodenum, trachea and lungs of mice in 3dpi in both mice strains. Antibodies against avian coronaviruses have been detected only in 3dpi. Thus, the results of this study demonstrate that the Massachusetts variant, originating from pigeon, cause clinical disease in commercial poultry unvaccinated and can replicate in mammalian model for a short period of time, emphasizing the importance of vaccination and the potential role of rodents as possible reservoir and the carrier virus
Doutorado
Microbiologia
Doutora em Genética e Biologia Molecular
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Davies, Marc Tudor. "Subcellular location and protein interactions of the infectious bronchitis virus gene 3 and 5 accessory proteins." Thesis, University of Warwick, 2009. http://wrap.warwick.ac.uk/3145/.
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The avian coronavirus infectious bronchitis virus (IBV) expresses four nonstructural, non-gene 1 proteins (3a, 3b, 5a and 5b) which have been shown to be dispensable for virus replication in cell culture. These IBV accessory proteins have no sequence homology to any of the accessory proteins of the group I and II coronaviruses but are highly conserved among the group III coronaviruses. Characterisation of naturally occurring strains of IBV which do not express two or more of the accessory proteins and of genetically modified recombinant IBVs has demonstrated that these accessory proteins contribute at most a minor role to the pathogenicity of the virus. To understand the relevance of these proteins for IBV the subcellular location of the 3a, 5a and 5b proteins have been characterised along with the identification of potential protein-protein interactions for the 3a protein. The subcellular location and protein-protein interactions of the 3b protein were attempted but specific problems were encountered. Indirect immunofluorescence with confocal microscopy of IBV-infected chick kidney cells was used to study the subcellular location of the accessory proteins. The 3a protein displayed a punctate, cytoplasmic distribution pattern which colocalised with virally-induced double-stranded RNA. A diffuse, cytoplasmic distribution was observed for the 5b protein which produced limited colocalisation with an IBV structural protein. Expression of a FLAG-tagged 5a protein in transfected Vero cells resulted in a punctate, cytoplasmic pattern. The protein interactions of the 3a protein were identified using FLAG-tag pull-down experiments with tandem mass spectrometry. Six cellular proteins were identified as interacting with the FLAG/3a protein within transfected Vero cells, three of which, GCN1, PP2A and Exportin-1, may interact with native 3a protein in IBV-infected cells. The 3a protein could sequester the viral dsRNA to hide it from the innate immune system and the potential interactions with three cellular proteins indicate that the IBV 3a protein may contribute to attenuation of host cell translation, induce cell cycle arrest and/or attenuate the nuclear export of a specific subset of mRNAs.
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Dalton, Kevin Paul. "Analysis of cis-acting signals required for the replication and packaging of infectious bronchitis virus defective-RNAs." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624435.
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Abd, el Rahman Sahar El Sayed El Sayed Ali [Verfasser]. "Comparative analysis of current infectious bronchitis virus isolates in primary cell culture systems / Sahar El Sayed El Sayed Ali Abd El Rahman." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2010. http://d-nb.info/1009660683/34.
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Forster, William Paul. "Risk, modernity and the H5N1 virus in action in Indonesia : a multi-sited study of the threats of avian and human pandemic influenza." Thesis, University of Sussex, 2012. http://sro.sussex.ac.uk/id/eprint/38647/.
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This thesis examines the Influenza A/H5N1 virus in action through an ethnographic study focused on the entwined concepts of risk and modernity. The objective is to explain why the response to the virus has been challenged in Indonesia. Concerned with policy formulation, and everyday practice, the thesis argues that assemblages of historical, political, institutional and knowledge‐power processes create multiple hybrid constructions of risk and modernity, which challenge technical responses based on epistemological positions and institutional arrangements that do not allow for such hybridity. The thesis is organised into four sections. The first section (chapters 1 – 3) introduces the virus and its terrain, outlines a constructivist position, and argues that conceptually risk and modernity have multiple, dynamic, power‐laden forms. The second section (chapters 4 – 6) contrasts constructions of risk and modernity among the actors and networks responding to the emergence, spread and persistence of the H5N1 virus, with the constructions of affected people in Indonesia. The third section (chapters 7 – 9) investigates the multi‐directional processes that occur when ‘global' policies and practices encounter ‘local' social and political settings, and vice versa, through three empirical case studies of the response to H5N1 in Indonesia between 2005 and 2010. The final section (chapter 10) provides a set of reflections and conclusions. Given the conceptual plurality of risk and modernity, and the multiple overlapping interacting hybrid constructions that have been empirically demonstrated in the case of H5N1, it is concluded that reductive, science‐based, governmentally‐orientated responses which treat nature as a matter of separate, fixed identity do not allow for such hybridity. The virus in action in Indonesia shows that any divide between nature and society is artificial and deceiving. Technical disease control responses need to incorporate understandings which accept the dynamics of culture, politics, and power.
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Okino, Cintia Hiromi. "Desenvolvimento da técnica de RT-PCR em tempo real para detecção e diferenciação de estirpes do vírus da bronquite infecciosa das galinhas /." Jaboticabal : [s.n.], 2007. http://hdl.handle.net/11449/95947.
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Orientador: Hélio José MontassierBanca: Clarice Weins Arns
Banca: Adolorata Aparecida Bianco Carvalho
Resumo: A bronquite infecciosa das galinhas (BIG) é uma doença infecciosa que está amplamente disseminada entre as criações avícolas brasileiras e é uma das enfermidades virais que mais têm causado perdas econômicas na atualidade. Portanto, a rápida detecção e identificação do agente causal são imprescindíveis para que medidas eficazes de controle sejam prontamente tomadas. Para tanto, é necessário que os métodos de diagnóstico empregados sejam sensíveis, específicos, rápidos e também de baixo custo. Nesse contexto, a técnica de RTPCR em tempo real abordada no presente estudo permitiu a amplificação de duas regiões de hipervariabilidade do gene S1 de 17 estirpes diferentes do vírus da BIG (VBI), que foram testadas, mas não foi capaz de amplificar nenhum dos RNAvírus heterólogos analisados (vírus da doença de Newcastle, pneumovírus aviário e vírus da doença de Gumboro). Com essa mesma técnica foi possível fazer a diferenciação em grupos geneticamente distintos, de estirpes do VBI através de análises das curvas de dissociação de fragmentos amplificados a partir das regiões de hipervariabilidade gênica I e II do gene S1. A RT-PCR em tempo real desenvolvida apresentou maior sensibilidade na detecção do VBI em amostras teciduais, quando comparada à técnica padrão de Isolamento Viral em ovos embrionados de galinha... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The avian infectious bronchitis virus (IBV) is an infectious disease widely spread in Brazilian commercial poultries where causes significant economical losses. Rapid and accurate diagnosis of the IBV strain involved in a field outbreak is necessary to establish an effective control of this disease. The real-time RT-PCR performed in this study to amplify two hypervariable regions of S1 gene, was able to detect 17 IBV strains, e.g., nine reference strains (including Massachussets, Connecticut, JMK, SE 17 and Iowa serotypes) and eight Brazilian field isolates, whilst non-related avian viral pathogens such as Newcastle disease virus, Avian Pneumovirus and Gumboro disease virus were not detected. The differentiation between IBV strains was accomplished using the melting curve analysis of the amplified fragments corresponding to the hypervariable regions I and II of S1 gene. The real-time RT-PCR developed here showed a higher rate of IBV detection in tissue samples of experimentally infected chickens, when compared to the goldstandard technique of Viral Isolation in embryonated chicken eggs, and the same rate of detection was found for the conventional RT-PCR... (Complete abstract, click electronic access below)
Mestre
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Piza, Vanessa Mirabelli Toledo [UNESP]. "Detecção e diferenciação do vírus da bronquite infecciosa pela técnica de imunocaptura-RT-PCR E RFLP." Universidade Estadual Paulista (UNESP), 2007. http://hdl.handle.net/11449/94869.
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piza_vmt_me_jabo.pdf: 496784 bytes, checksum: 8e82f3b02d746dfe3daa52fd23b8be9e (MD5)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Nesse estudo foi desenvolvido e aplicado o procedimento de imunocaptura para realizar a reação de transcrição reversa seguida pela reação em cadeia da polimerase (IC-RT-PCR) a fim de ser amplificada uma região 5'_ proximal do gene 81 do vírus da bronquite infecciosa (VBI) com 228 pb e de forma a fazer a detecção e a diferenciação desse vírus, comparando-se os resultados dessa metodologia com os obtidos na técnica convencional de RT-PCR. Todas as 11 estirpes do VBI testadas foram amplificadas pelas duas técnicas moleculares, enquanto que nenhum dos vírus heterólogos (Pneumovírus Aviário do grupo A e B, Vírus da Doença de Gumboro e o Vírus da Doença de Newcastle) ensaiados levaram a amplificação específica do gene 81. O limiar de detecção para a técnica de IC-RT-PCR foi idêntico ao do método de RT-PCR e correspondeu a 102.8 Doses Infectantes Embrionárias 50%. Para um total de 35 amostras de tecidos do trato respiratório testadas e provenientes de aves infectadas experimentalmente, 32 foram positivas pela técnica de IC-RT-PCR e também pela RT-PCR convencional, enquanto que o isolamento viral foi obtido para 22 dessas amostras. A análise do produto amplificado do gene 81 na IC-RT-PCR através da técnica de RFLP (restriction fragment length polymorphism), com as enzimas Alul e Mboll, permitiu a diferenciação das 5 estirpes de referências e dos 6 isolados de campo analisados em 4 diferentes genótipos, correspondentes às estirpes de referência M41, Connecticut, ou 8E-17, ou a um isolado de campo. Portanto, a técnica de IC¬RT -PCR demonstrou um grande potencial para ser aplicada no diagnóstico direto do VBI, apresentando vantagens sobre a técnica convencional de RT-PCR, as quais foram derivadas da combinação da especificidade da imunocaptura em fase sólida com a sensibilidade da reação de PCR, o que pode proporcionar ganho de tempo e menor custo para a manipulação de um maior número de amostras.
In this study, the immunocapture procedure followed by reverse transcription and polymerase chain reaction (IC-RT-PCR) technique was standardized and applied for the amplification of 5'- proximal pari of 81 gene of infectious bronchitis virus (IBV) in infected f1uid or tissue samples, which were collected from embryonating chicken eggs or experimentally infected birds. The results of this technique were compared with those obtained in conventional RT-PCR. Ali eleven IBV strains tested were amplified, while none of the heterologous avian viral pathogens (Groups A and B Avian Pneumovirus, Newcastle Disease Virus and Gumboro Disease Vírus) gave positive results. The limit of detection for IC-RT¬PCR was identical to the common RT-PCR and corresponded to 102.8 50% embryonic infectious doses. Thirty two out of thirty five respiratory tissue samples collected from experimentally infected chickens were positive by both molecular techniques (IC-RT-PCR I common RT-PCR), whereas the vírus isolation test detected IBV in twenty two of these samples. The AluL and Mobll RFLP analysis of the 228 bp amplicon generated from IC-RT-PCR led to the discrimination of the 5 reference strains and 6 field IBV isolates in four genotypes; which were associated to the M41, to Connecticut, ar to 8E-17 strain, or to a field isolate. Therefore, the IC-RT-PCR demonstrated in this study a high potential for the application in the direct diagnosis of IBV and has relevant advantages over the conventional RT¬PCR, because it combines the specificity of the immunocapture in a solid phase with the sensitivity of the PCR, providing simplicity, rapidity and low cost for the manipulation of a high number of samples.
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Caetano, Aline Gonçalves. "Produção dos fragmentos de anticorpos recombinantes scFv-N e scFv-S1 e suas aplicações na detecção e diferenciação do Vírus da Bronquite Infecciosa /." Jaboticabal : [s.n.], 2009. http://hdl.handle.net/11449/103885.
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Orientador: Hélio José MontassierBanca: João Pessoa Araujo Júnior
Banca: Valéria Marçal Félix de Lima
Banca: Edison Luiz Durigon
Banca: Jesus Aparecido Ferro
Resumo: O vírus da Bronquite infecciosa (VBI) é um Coronavírus aviário que infecta aves domésticas de corte e postura, ocasionando grandes perdas econômicas na indústria avícola. Dada a natureza altamente contagiosa e aguda da doença, há uma grande necessidade do desenvolvimento de métodos diagnósticos que possam ajudar na detecção e/ou caracterização de estirpes variantes do VBI. Sendo assim, para auxiliar no diagnostico laboratorial da infecção, foi construída uma biblioteca de fragmentos de anticorpos monoclonais pela técnica de Phage-display. Para tanto, após a imunização de galinhas com a estirpe vacinal H120, foi extraído o RNA total do baço das aves imunizadas e amplificadas as cadeias variáveis leve e pesada que foram unidas por linker, originando o fragmento gênico de cadeia única scFv. Após a realização de três ciclos de seleção foram obtidos 400 clones que foram avaliados em ensaios de ELISA e Western blotting para averiguação da especificidade dos mesmos frente às proteínas da estirpe H120. Após realização dos testes foram selecionados dois clones, um que apresentou grande reatividade para com a proteína de nucleocapsídeo (N) (scFv-N) e o outro com reatividade para com a subunidade 1 da glicoproteína de superfície (S) (scFv-S1). O anticorpo scFv-S1 quando utilizado em ensaio de vírus-neutralização em ovos embrionados mostrou titulo significativo de proteção. Já em testes de ELISA utilizando estirpes de referência e isolados brasileiros de campo do VBI, o anticorpo scFv-N foi capaz de detectar todas as estirpes (H120, M41, Arkansas, IBVPR05, IBVPR02, IBVPR01, IBVSC01), enquanto que o scFv-S1 pode discriminar as estirpes pertencentes ao sorotipo Massachusetts (H120, M41 e IBVSC01) das demais estirpes variantes avaliadas. Os fragmentos de anticorpos scFv-N e scFv-S1 também mostraram bons resultados quando utilizados na técnica... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Infectious bronchitis virus (IBV), the coronavirus of the chicken, is one of the main causes of economic loss within the poultry industry, affecting the performance of meattype and egg-laying domestic fowls. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect and/or characterize IBV strains. In order to improve the laboratory diagnosis of IBV infection, phage-displayed recombinant antibody library derived from splenic mRNA of chickens immunized with H120 vaccine strain of infectious bronchitis virus (IBV) was constructed as single chain variable fragments (scFv) by overlap extension polymerase chain reaction (PCR) of the individual heavy (VH) and light (VL) chain variable gene segments. After three rounds of panning selection, ten scFv phage display antibodies of 400 randomly chosen clones were demonstrated to react with IBV antigens by ELISA. The western blot analysis selected two scFv antibodies reacting strongly with nucleocapsid (N) (scFv-N) protein or subunit 1 of spike glycoprotein (S1) (scFv-S1) of IBV. The anti-S1 scFv antibody showed a significant neutralization titre in embryonating chicken egg test. In ELISA analysis using reference IBV strains and Brazilian field isolates, the anti-N scFv antibody was able to detect all strains (H120, M41, ARKANSAS, IBVPR05, IBVPR02, IBVPR01, IBVSC01), while the anti-S1 could discriminate Massachusetts serotype (H120, M41 and IBVSC01) between variant strains. A scFv-based indirect immunoperoxidase (IP) procedure was also applied to detect infectious bronchitis virus (IBV) antigens in formalin-fixed tracheal tissue sections. Thus, the results showed that scFv-N and scFv-S1 antibodies can be used for the detection and differentiation of IBV strains.
Doutor
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Gibertoni, Aliandra Maura [UNESP]. "Clonagem e expressão do gene da nucleoproteína do vírus da bronquite infecciosa em sistemas hospedeiros eucarioto (Pichia pastoris) e procarioto (Escherichia coli)." Universidade Estadual Paulista (UNESP), 2009. http://hdl.handle.net/11449/103926.
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Foram realizadas a clonagem e expressão do gene da nucleoproteína (N) de uma estirpe vacinal de referência M41 do vírus da bronquite infecciosa (VBI), como proteína recombinante de fusão, contendo uma cauda de poli-histidina na extremidade carboxi-terminal, em 2 sistemas hospedeiros; na levedura metilotrófica, Pichia pastoris e na bactéria Escherichia coli. A proteína N derivada de um isolado variante do VBI de surtos a campo no Brasil, também foi expressa em E. coli. As características bioquímicas e imunoquímicas de tais proteínas recombinantes, foram determinadas, tendo sido evidenciado maior eficiência de produção no sistema hospedeiro constituído por E. coli, comparativamente ao sistema composto por P. pastoris. Uma vez obtidas, caracterizadas e purificadas, através da técnica de cromatografia de afinidade em resina de níquel-sepharose, as preparações de proteína N recombinante expressas em E. coli e derivadas ou da estirpe de referência M41 ou do novo isolado de campo no Brasil, foram utilizadas de forma bem sucedida, como antígenos alvo de ensaios indiretos de ELISA, que foram aplicados na detecção e mensuração de anticorpos dos isótipos IgG e IgM em aves infectadas com estirpes homóloga ou variantes do VBI. Foi, também, investigada a atividade imunogênica da proteína N recombinante em aves, que depois de imunizadas e re-imunizadas com essas proteínas recombinantes, produziram no soro sanguíneo e na secreção lacrimal quantidades elevadas de anticorpos anti-VBI específicos, mas não desenvolveram proteção efetiva contra o desafio com a estirpe homóloga desse vírus. Concluindo, a proteína N recombinante do VBI expressa pela E. coli possui elevada imunogenicidade, no sentido de induzir altos níveis de anticorpos específicos, e reatividade cruzada com proteínas N de outras variantes desse vírus, tendo um grande potencial de ser aplicada em...
Two host systems, represented by Escherichia coli and Pichia pastoris were used for cloning and protein expression of the nucleoprotein (N) gene of M41 strain of infectious bronchitis virus (IBV) as a fusion recombinant protein containing a poli-histidine tag. The N protein from a new variant Brazilian field isolate was also cloned and expressed by E. coli system. The biochemical and immunochemical properties of these recombinant N proteins were determined and higher efficiency on protein production was achieved by using the E. coli expression system. Both recombinant N proteins expressed by E. coli were purified in nickel-sepharose resin and used as antigen in indirect ELISA methods for the detection of IgG and IgM antibodies in birds infected with homologous and variant IBVs. The immunogenicity of N recombinant protein was also evaluated by immunizing and re-immunizing birds and high antibody levels were generated in lachrymal secretion and serum, but no effective protection against challenge with homologous virulent stain of IBV was induced. Concluding, the recombinant N IBV protein expressed by E. coli is highly immunogenic for inducing specific and crossreactive antibodies, and can be applied in the immuno-diagnosis of IB
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Okino, Cintia Hiromi [UNESP]. "Desenvolvimento da técnica de RT-PCR em tempo real para detecção e diferenciação de estirpes do vírus da bronquite infecciosa das galinhas." Universidade Estadual Paulista (UNESP), 2007. http://hdl.handle.net/11449/95947.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A bronquite infecciosa das galinhas (BIG) é uma doença infecciosa que está amplamente disseminada entre as criações avícolas brasileiras e é uma das enfermidades virais que mais têm causado perdas econômicas na atualidade. Portanto, a rápida detecção e identificação do agente causal são imprescindíveis para que medidas eficazes de controle sejam prontamente tomadas. Para tanto, é necessário que os métodos de diagnóstico empregados sejam sensíveis, específicos, rápidos e também de baixo custo. Nesse contexto, a técnica de RTPCR em tempo real abordada no presente estudo permitiu a amplificação de duas regiões de hipervariabilidade do gene S1 de 17 estirpes diferentes do vírus da BIG (VBI), que foram testadas, mas não foi capaz de amplificar nenhum dos RNAvírus heterólogos analisados (vírus da doença de Newcastle, pneumovírus aviário e vírus da doença de Gumboro). Com essa mesma técnica foi possível fazer a diferenciação em grupos geneticamente distintos, de estirpes do VBI através de análises das curvas de dissociação de fragmentos amplificados a partir das regiões de hipervariabilidade gênica I e II do gene S1. A RT-PCR em tempo real desenvolvida apresentou maior sensibilidade na detecção do VBI em amostras teciduais, quando comparada à técnica padrão de Isolamento Viral em ovos embrionados de galinha...
The avian infectious bronchitis virus (IBV) is an infectious disease widely spread in Brazilian commercial poultries where causes significant economical losses. Rapid and accurate diagnosis of the IBV strain involved in a field outbreak is necessary to establish an effective control of this disease. The real-time RT-PCR performed in this study to amplify two hypervariable regions of S1 gene, was able to detect 17 IBV strains, e.g., nine reference strains (including Massachussets, Connecticut, JMK, SE 17 and Iowa serotypes) and eight Brazilian field isolates, whilst non-related avian viral pathogens such as Newcastle disease virus, Avian Pneumovirus and Gumboro disease virus were not detected. The differentiation between IBV strains was accomplished using the melting curve analysis of the amplified fragments corresponding to the hypervariable regions I and II of S1 gene. The real-time RT-PCR developed here showed a higher rate of IBV detection in tissue samples of experimentally infected chickens, when compared to the goldstandard technique of Viral Isolation in embryonated chicken eggs, and the same rate of detection was found for the conventional RT-PCR... (Complete abstract, click electronic access below)